Xanthomonas oryzae pv.oryzicola（Xoc） causes a destructive bacterial leaf streak disease in rice.Some of the gene products annotated as hypothetical proteins in the genome of Xoc may contribute to its virulence in rice.A mutant,Mxoc1679,screened from our previous Tn5-tagged mutant library for Xoc strain RS105,showed reduced virulence in rice.In this mutant,a gene named as Xoryp_08180 was disrupted by Tn5 insertion.Xoryp_08180 encodes a 1 306-aa hypothetical protein which is highly conserved in Xanthomonas spp.Non-polar mutation of Xoryp_08180 in RS105 strain led to a significant reduction in bacterial virulence and growth in rice,a delayed hypersensitive response（HR） in non-host tobacco,and a decrease in extracellular protease activity.The deficiencies above were restored to wild-type level in the complementary strain by expressing Xoryp_08180 in trans.In addition,the expression of Xoryp_08180 was repressed in hrpG and hrpX mutants in planta but not in a nutrient-rich condition.These results suggested that Xoryp_08180 is a virulence factor required for extracellular protease production,HR induction and full virulence of Xoc.
Xanthomonas oryzae pv.oryzicola （Xoc）,the critical pathogen causing bacterial leaf streak in rice,possesses a hrp cluster that is responsible for triggering hypersensitive response （HR） in non-host tobacco and pathogenicity in host rice,and is considered to be one of the model pathogens in the rice model plant.Here,we developed a high-throughput mutagenesis system using a two-step integration mediated by a novel suicide vector pKMS1.It was used to generate single or poly-gene mutants of hpa1,hpa2,hrcV,hrpE,hpaB,and hrpF gene for functional analysis.In total,five single,four double,and two triple hrp gene mutants were constructed.The double and triple hrp gene deletion mutants triggered novel phenotypes in planta.Our data suggest that pKMS1 is a useful tool for non-marker mutagenesis of multiple genes in Xoc.
